Corneal opacities, a leading cause of blindness worldwide, are commonly a result of stromal fibrosis or haze from dysregulated wound healing. Corneal wound healing is an enormously complex process that requires the simultaneous cellular integration of multiple soluble biochemical as well as biophysical cues associated with the wound space. Research from our laboratory and others have demonstrated biophysical attributes of the extracellular matrix profoundly modulate a host of fundamental cellular behaviors essential to the maintenance of homeostasis and wound repair including adhesion, migration, proliferation and differentiation. We have demonstrated that the intrinsic mechanical properties of the corneal stroma are significantly altered in a time dependent manner throughout wound healing. The matrix associated with the wound space was stiffest at 7 days with the greatest myofibroblast numbers and degree of stromal haze occurring at a later time point. This temporal relationship, combined with in vitro data documenting stiffer substrates to promote myofibroblast transformation, suggests a causal relationship. In Aim 1 we propose to directly modulate matrix stiffness in vivo using clinically relevant approaches and establish the impact of stiffening and softening of the wound matrix on wound healing outcomes using a well-defined rabbit PTK model. The complex inter-relationship between cytoskeletal dynamics and matrix remodeling and stiffness is central to determining wound healing outcomes but remains little investigated and poorly understood and are the central focus of Aim 2. Here, the impact of altering cytoskeletal dynamics in vitro on cell derived extracellular matrix elaboration, remodeling and mechanics, and the impact of altering cell derived matrix stiffness on keratocytes to myofibroblast transformation will be determined. Preliminary data demonstrate that the mechanical properties of ECM derived from cell in vitro can be modulated by applying diverse clinically relevant crosslinking methods. Preliminary data also document that modulating the cellular stiffness using drugs that alter the cytoskeleton can dramatically influence myofibroblast transformation. Stiffening of the cell promoted myofibroblast transformation. While softening of the cell inhibited transformation even in the presence of TGF? signaling. Additionally, the complex signaling dynamic that exist between stromal and epithelial cells will be investigated using a novel microfluidic co-culture platform in Aim 3. This novel system allows for the simultaneous presentation of multiple yet distinct biophysical and biochemical stimuli in addition to controlled exchange of soluble signaling molecules between epithelial and stromal cells. We will determine if soluble signals from the epithelial cells have a differential effect on KFM transformation under various biophysical stimuli. In aggregate the experiments detailed in this proposal are aimed at testing our central concept that optimal outcomes in corneal wound repair can be facilitated by engineering the intrinsic biophysical attributes of cells and/or matrices.